Anti-hiv composition, production method thereof and medicament

ABSTRACT

The present invention relates to an anti-HIV composition and to the method for producing it. The composition of the present invention comprises a polyanion and a molecule capable of inducing the exposure of the CD4i epitope of the gp120 viral protein. The polyanion may be chosen, for example, from the group consisting of heparin, heparan sulphate, and a polyanion equivalent to heparin or to heparan sulphate. The molecule capable of inducing the exposure of the CD4i epitope of the gp120 viral protein is a CD4 peptide or a derivative thereof. The present invention also relates to the use of said composition for producing a medicinal product, in particular a medicinal product intended for the treatment of AIDS.

TECHNICAL FIELD

The present invention relates to an anti-HIV composition and to themethod for producing it. It also relates to the use of said compositionfor producing an anti-HIV medicinal product.

Entry of the human immunodeficiency virus (HIV) into the cell is anessential step in the viral infectious cycle. This process is divided upinto two phases corresponding to the interaction of the virus at thecell surface at the level of specific receptors of the host, and to thepenetration of the genetic material of the virus into the target cell.

Over the past ten years, the mechanisms of adhesion of HIV to the cellsurface have become considerably clarified. The molecular partnersinvolved are now well defined, as disclosed in documents [1, 2] of thereference list attached in the appendix.

As regards the virus, the envelope glycoproteins gp120 and gp41constitute the “key to the vault” from the virus/cell interactioncomplex. Initially, g-120 associates with a transmembrane protein of thehost cell, CD4. This interaction results in a conformational change ingp120, which will expose a particular epitope, referred to as“CD4-induced” epitope (CD4i). CD4i constitutes a binding site forcertain members of the chemokine receptor family (mainly CXCR4 andCCR5), which will play a role of gp120 coreceptor at the cell surface.This second interaction, gp120/CCR5 or gp120/CXCR4, then results in areorganization of the gp120/gp41 protein complex. This reorganisationexposes gp41, which then allows initiation of the fusion of the cell andviral membranes, and entry of the viral genetic material into the cell.

These studies make it possible to define two novel therapeutic targets:inhibition of the interaction of gp120 with CD4 and CCR5 or CXCR4, andinhibition of the fusion [3].

PRIOR ART

The references between [ ] refer to the reference list attached in theappendix.

In the field of human immunodeficiency virus (HIV) infection,tritherapies associating nucleoside inhibitors, non-nucleosideinhibitors and/or antiproteases (“HAART” for “Highly ActiveAntiretroviral Treatment”) target the replication and maturation of thevirus.

These treatments make it possible to substantially reduce the viralload, but they do not make it possible to totally eradicate the virusfrom the body. In fact, if the taking of medicinal products is stopped,even after several years of treatment, this invariably results in arapid increase again in the viral load in the plasma. Besides thisdisadvantage, these treatments are considerably toxic and have many sideeffects.

In the context of the search for new treatments against AIDS, theprocesses of adsorption of the virus onto the host cell constitute aparticularly attractive therapeutic target due in particular to the factthat this step takes place outside the cell.

Peptides which bind to gp41 and which inhibit its fusion activity havebeen developed [4, 5]. The clinical studies currently in progress givepositive results, indicating that inhibition of the fusion, andtherefore of the entry of the virus, effectively corresponds to anadvantageous therapeutic target.

As regards the attachment of the virus, various studies have exploredthe use of soluble CD4 for inhibiting the interaction of the virus withthe CD4 expressed at the surface of cells that are targets for HIV. Thissolution has proved to be ineffective, because, in binding to the virus,the soluble CD4 exposes the CD4i epitope and in fact promotes theinteraction of the virus with the CCR5 or CXCR4 coreceptor, which, incertain cases, increases infection [6].

In addition to CD4, the coreceptors are also sites of attachment of thevirus to the cells. The natural ligands for these coreceptors arechemikines, in particular RANTES and MIP for CCR5, and SDF for CXCR4. Invitro or on cells in culture, these chemokines inhibit the interactionof the virus with the cells [7, 8], but also induce a certain number ofcell responses making them difficult to use from a therapeutic point ofview. A certain number of compounds such as AMD301 or peptides whichbind to the coreceptors also have antiviral effects [9, 10]. However, intargeting the HIV coreceptors, these various molecules also block theintrinsic functions of the cell linked to the use of these coreceptors.

Besides these cell receptors, HIV is capable of binding to othermolecules present on the cells that it infects, such as DC-SIGN,sphingolipids or heparan sulphates [11].

Heparan sulphates are complex polysaccharides belonging to theglycosaminoglycan (GAG) family. They are abundantly present at the cellsurface and in interstitial matrices, where they are found anchored tothe extracellular domain of specific glycoproteins, heparan sulphateproteoglycans (HSPGs). Heparan sulphates (HSs), which were discoveredhalf a century ago from preparations of heparin (another type of GAGhaving very similar properties), differ from any other biologicalmacromolecule by virtue of the diversity of their structure and of thefunctions that they exercise. They are capable in particular of bindingHIV gp120, and the virus uses this property in order to adsorb to thesurface of target cells. The site of interaction of heparan sulphates ongp120 is located on a variable structure, called V3 loop [12]. However,the exact role of these polysaccharides during infection with HIVremains relatively unclear. Studies have shown that elimination of theheparan sulphates expressed at the surface of cells contributes tomaking them less permissive to infection with the virus [11],demonstrating the importance of this molecule for the attachment and theentry of the virus.

On the basis of these observations, various polyanionic molecules of theheparin type have been developed in order to inhibit the interaction ofthe virus with the cells. However, the first clinical trials have shownonly little or no activity of these molecules, and it has been possibleto observe toxic effects in certain cases [13, 14].

It therefore appears to be necessary to develop new treatments againstAIDS that are less restricting, result in fewer side effects and make itpossible to avoid evasion of the treatment, i.e. the appearance ofresistant viruses which no longer respond to the treatments. It is alsonecessary to find other anti-HIV therapies that are directed against newtargets.

It is in this context that the inventors have produced the presentinvention.

DISCLOSURE OF THE INVENTION

The aim of the present invention is precisely to overcome theabovementioned disadvantages by providing a novel composition which canbe used as an anti-HIV agent. This composition is capable of blockingthe entry of the AIDS virus into its host cells. In this respect, it canbe used for preparing a medicinal product, in particular a medicinalproduct intended for the treatment of AIDS.

The composition of the present invention is characterized in that itcomprises a polyanion and a molecule capable of inducing the exposure ofthe CD4i epitope of the gp120 viral protein.

Thus, in accordance with the present invention, the inventors havecombined, within a single composition, firstly, a polyanion, for exampleof the heparin or heparan sulphate type and, secondly, a moleculecapable of inducing the exposure of the CD4i epitope of the gp120 viralprotein, for example of a soluble CD4 peptide. They have shown that thiscomposition makes it possible to inhibit, unexpectedly, both thevirus-cell membrane heparan sulphate interaction, by blocking the V3loop, and the virus-coreceptor interaction, by blocking the CD4i site.In fact, the inventors have shown that there are actually two domains orsites of interaction of heparin- or heparan sulphate-polyanions ongp120. The first is the V3 loop, the second is the CD4i domain. Theyhave shown (see examples below) that heparin, or heparin fragments ofsufficient size, in the presence of a CD4 peptide, interacts with theCD4i domain of the gp120 viral protein and that this combination greatlyinhibits the gp120/48d or 17b antibody interaction. 48d or 17b are usedas mimics of coreceptors.

This blocking of HIV with the composition of the present invention isall the more unexpected since those skilled in the art are aware thatthe CD4 molecule used alone can have the reverse effect of that desired,since it exposes the domains for interaction with the coreceptors, andcan therefore increase the virus infectivity.

The composition of the present invention is therefore directed towards anovel therapeutic target by means of heparin or other polyanions in thepresence of the CD4 peptide, namely the blocking of the interaction ofHIV with its coreceptors. This solution is very advantageous, from thetherapeutic point of view, for inhibiting the attachment of the virus tothe cells, since it targets the virus itself and not the cells. It istherefore, firstly, free of the cellular effects which are observed withthe products of the prior art when it is the coreceptors that aretargeted. In addition, the toxicity of the composition of the presentinvention for an organism is less than most of the chemical compounds ofthe prior art due to the nature of its constituents.

According to the invention, the polyanion may advantageously be chosenfrom the group consisting of heparin, heparan sulphate, and a polyanionequivalent to heparin or to heparan sulphate. It is, for example,Dextran sulphate (commercial name, Ueno fine chem), Curdlan sulphate(commercial name, Ajinomoto), 2-Naphthalenesulphonate polymer(commercial name, Procept), Pentosan polysulphate (commercial name,Baker norton pharm; Hoechst), or Resobene (commercial name).

The structure of the constituent disaccharide (basic element) of theheparin and of the heparan sulphate according to the present inventionis of formula (I) below:

It is preferable for the polyanion not to be too long, since it wouldhave an anticoagulant activity, which is not desired in the presentinvention, and would form aspecific bonds with various proteins, inparticular thrombin or antithrombin III. Its length will preferably besimilar to a heparin chain having a degree of polymerization as definedbelow. The polyanion preferably has at least two anionic groups perdisaccharide. According to the present invention, when the polyanion isheparin or heparan sulphate, it will preferably have a degree ofpolymerization dp of 10 to 24, advantageously of 12 to 24, preferably of16 to 22. According to the invention, the heparin, the heparan sulphateor the polyanion equivalent to heparin or heparan sulphate may have adegree of polymerization dp of 12 to 20, for example of 15 to 17.

According to the invention, the polyanion may be prepared by partialdepolymerization of heparin or of heparan sulphate by means of anenzymatic method, for example by means of heparinase, or a chemicalmethod, for example by means of nitrous acid. When they are obtainedchemically, the heparans may be defined by the presence of N-sulphatedor N-acetylated glucosamine, or glucosamine not substituted in theN-position, linked to a uronic acid (glucuronic acid or iduronic acid)with a variable proportion of sulphate group. Structural mimics of theseoligosaccharides may be obtained by chemical synthesis.

According to the invention, the molecule capable of inducing theexposure of the CD4i epitope of the gp120 viral protein can be chosenfrom a CD4 peptide or a derivative of this peptide, or else a monoclonalantibody which binds to the gp120 viral protein and which is capable ofactivating said gp120 protein in a manner equivalent to the CD4 peptide.

When it is a CD4 peptide, it is preferably soluble for the obviousreasons of facilitation of its interaction with the gp120 viral proteinin liquid medium, and of facilitation of its access to its target.

According to the invention, the CD4 peptide advantageously has thesequence (I) below:

Cys or TPA-P¹-Cys-P²-Cys-P³-Cys-Ala or Gln-Gly or (D)Asp or Ser-Ser orHis or Asn-Xaa^(J)-Cys-Thr or Ala-Cys-Xaa^(k)-NH₂

in which TPA represents thiopropionic acid, Xaa^(J) representsβ-naphthylalanine, phenylalanine or biphenylalanine, Xaa^(k) representsGly, Val or Ileu, P¹ represents 3 to 6 ammo acids, P² represents 2 to 4amino acids and P³ represents 6 to 10 amino acids, the amino acids inP¹, P² and P³ being natural or unnatural, identical or different, andP¹, P² and P³ possibly having a common sequence, said peptide having aβ-hairpin conformation in which the β-turn is made up of the amino acidresidues Ala or Gln-Gly or DAsp or Ser-Ser or His or Asn-Xaa^(J) of itssequence (A). In fact, these peptides show a very great affinity for thegp120 viral protein.

Examples of such peptides which can be used in accordance with thepresent invention are the peptides of sequences ID No. 1 to ID No. 18 ofthe sequence listing attached in the appendix, or equivalent peptides.

These peptides can be prepared by conventional techniques of solid-phasechemical synthesis or of genetic recombination.

When the molecule capable of inducing the exposure of the CD4i epitopeof the gp120 viral protein is an antibody, it can be chosen, forexample, from those described in the document Sullivan N, Sun Y, BinleyJ, Lee J, Barbas C F 3rd, Parren P W, Burton D R, Sodroski J,Determinants of human immunodeficiency virus type 1 envelopeglycoprotein activation by soluble CD4 and monoclonal antibodies. JVirol 1998; 72(8): pp. 6332-6338.

According to a first embodiment of the present invention, the polyanionand the molecule capable of inducing the exposure of the CD4i epitope ofthe gp120 viral protein are mixed in said composition. This compositionin accordance with the present invention makes it possible to expose thesite of interaction with the coreceptors (CD4i site) and, concomitantly,to block this site by means of the oligosaccharide part consisting ofthe polyanion.

According to this first embodiment, the polyanion and the moleculecapable of inducing the exposure of this CD4i epitope of the gp120 viralprotein are advantageously mixed in said composition in proportions of 1to 10 mol of polyanion per 0.5 to 1.5 mol of molecule capable ofinducing the exposure of the CD4i epitope of the gp120 viral protein,preferably of 5 mol of polyanion per mole of molecule capable ofinducing the exposure of the CD4i epitope of the gp120 viral protein.

The present invention also relates to a method for producing thecomposition according to this first embodiment of the invention,comprising the following steps:

preparing the polyanion,

preparing the molecule capable of inducing the exposure of the CD4iepitope of the gp120 viral protein,

mixing the polyanion and the molecule capable of inducing the exposureof the CD4i epitope of the gp120 viral protein prepared so as to obtainsaid composition.

The mixture will preferably be prepared in a biological buffer so thatit can be used to produce an administrable medicinal product. The pH ispreferably approximately 7, and the solution contains, for example, 15g/l of NaCl.

According to a second embodiment of the present invention, the polyanionand the molecule capable of inducing the exposure of the CD4i epitope ofthe gp120 viral protein are linked to one another in said composition.They form a hybrid of polyanion/molecule capable of inducing theexposure of the CD4i epitope of the gp120 viral protein hybrid.

For example, according to the invention, the polyanion and the moleculecapable of inducing the exposure of the CD4i epitope of the gp120 viralprotein are linked to one another at one of the ends of the polyanion.

When the polyanion used is short, for example with a degree ofpolymerization dp of 10 to 12, it may be necessary to link the polyanionand the molecule capable of inducing the exposure of the CD4i epitope ofthe gp120 viral protein by means of a spacer arm, in order to allow thehybrid formed to bind to all its targets on the gp120 viral protein.This may also be the case when the molecule capable of inducing theexposure of the CD4i epitope of the gp120 viral protein is too short.The spacer arm may be any polymer, preferably soluble in aqueousbuffers, of appropriate length. Mention may be made, for example, ofpolyosides or polyglycols. It may be, for example, polyethylene glycol:(CH₂CH₂O)_(n). Preparations of spacer arms of this type which can beused in the present invention have been widely described in the priorart, for example in documents [18] and [19] (see attached referencelist).

The present invention also relates to a method for producing thecomposition according to the second embodiment of the invention,comprising the following steps:

preparing the polyanion,

preparing the molecule capable of inducing the exposure of the CD4iepitope of the gp120 viral protein,

linking the polyanion and the molecule capable of inducing the exposureof the CD4i epitope of the gp120 viral protein prepared so as to obtainsaid composition.

The linking of the polyanion with the molecule capable of inducing theexposure of the CD4i epitope can be formed by any techniques known tothose skilled in the art, for example for linking a polyanion and apeptide. For example, the various methods described in documents [15],[16] and [17] (see attached reference list) for coupling anoligosaccharide to a polypeptide can be used in the present invention.

According to the present invention, for the reasons mentioned above, itis also possible to use any type of bridging agent, or spacer arm, whichbinds, firstly, to one end of the oligosaccharide and, secondly, to apart of CD4 that is not essential to its function. The spacer arm may beone of those mentioned above. It can be prepared in the manner describedin documents [18] and [19].

The hybrid molecule of the present invention has three advantages: itbinds to the gp120 viral protein on the CD4 interaction site, on the V3loop, when these gp120 molecules are derived from viruses using CXCR4 ascoreceptor, and on the domain of interaction with the coreceptors (CD4idomain), as shown diagrammatically in FIG. 7 attached in the appendix.It therefore makes it possible to simultaneously block all the domainsthat gp120 uses to interact with its cell receptors and coreceptors.

Other characteristics and advantages will become apparent to thoseskilled in the art in the light of the examples below, given by way ofnon-limiting illustration, with references to the figures and sequencesattached in the appendix.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The sequences ID No. 1 to ID No. 18 of the sequence listing attached inthe appendix are non-limiting examples of molecules capable of inducingthe exposure of the CD4i epitope of the gp120 viral protein for thepurpose of the present invention. These molecules are peptidesoriginating from human CD4 (Seq ID No. 1), or artificial peptidesoriginating or derived from scorpion venom peptides (Seq ID Nos. 2 to18).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph representing the amount of gp120/CD4, in resonanceunits (RU), bound to heparin as a function of time t (in seconds) forvarious concentrations of CD4 (in nM): curves from bottom to top: 0; 50;100; 250; 500 nM.

FIG. 2 is a graph representing the amount of gp120/CD4, in resonanceunits (RU), bound to heparin as a function of the concentration of CD4(in nM).

FIG. 3 is a graph representing the evolution of the gp120 viralprotein/antibody 48d interaction (response RU) as a function of time (inseconds) for various concentrations of CD4 peptide (in nM): curves frombottom to top: 0; 50; 100; 250; 500 nM.

FIG. 4 is a graph representing the inhibition of the interaction of thegp120/CD4 complex with the antibody 48d (response RU) as a function oftime (in seconds) by various concentrations of heparin H (in nM): curvesfrom top to bottom: 0; 3; 6; 12; 30 μg/ml.

FIG. 5 is a graph representing the inhibition of the amount of gp120/CD4complexes bound to 48d as a function of the size of the heparin fragmentin degree of polymerization, from dp 0 to dp 18 (curves from top tobottom: dp 0; dp 2; dp 4; dp 6; dp 8; dp 10; dp 12; dp 14; dp 16; dp18).

FIG. 6 is a graph representing the amount of gp120/CD4 complexes boundto 48d as a function of the size of the heparin fragment based on thedata represented in FIG. 5.

FIG. 7 is a drawing showing diagrammatically the gp120 viral protein,and the interaction of a composition in accordance with the secondembodiment of the present invention with the gp120 viral protein. Inthis figure, sCD4bs (“s” for soluble, “bs” for binding site)=CD4-bindingsite, V3=V3 loop, CD4ni=non-induced (“ni”), (non-accessible)coreceptor-binding site, CD4i=coreceptor-binding site induced by thebinding of CD4 to gp120 (“i” for induced), CD4/H=hybrid molecule of CD4peptide/heparin or heparan sulphate according to the second embodimentof the present invention, and H=heparin or heparan sulphate.

FIGS. 8 a) to h) are representations in the form of graphs of theinteraction of complexes of gp120 viral protein and gp120/CD4 on anactivated sensorchip with heparin, at various concentrations of gp120viral protein: 0 nM (a); 0.62 nM (b); 1.25 nM (c); 2.5 nM (d); 5 nM (e);10 nM (f), 20 nM (g) and 40 nM (h), with preincubation (continuouslines) or without preincubation (discontinuous lines) with 80 nM ofsoluble CD4.

FIG. 9 is a representation in the form of a graph of the inhibition ofthe gp120/CD4 interaction by heparin and oligosaccharides of heparin (H)on a sensorchip activated with mAb17b. The gp120 viral protein (5 nM)was preincubated successively with a CD4 peptide (10 nM) and withconcentrations of heparin ([H]) at 0 nM (curve a); 2.1 nM (b); 4.2 nM(c); 8.3 nM (d); and 16.7 nM (e), before being injected onto the mAb17bsurface.

FIG. 10 is a representation in the form of a graph of the inhibition ofthe gp120/CD4 interaction by heparin and oligosaccharides of heparin (H)on the sensorchip activated with mAb17b. The gp120 viral protein (5 nM)was preincubated successively with a CD4 peptide (10 nM) and with aconcentration of heparin of 40 nM, with various degrees ofpolymerization: dp 0 (curve 1); dp 2 (curve 2); dp 4 (curve 3); dp 6(curve 4); dp 8 (curve 5); dp 10 (curve 6); dp 12 (curve 7), dp 14(curve 8); dp 16 (curve 9); dp 18 (curve 10), before being injected ontothe mAb17b surface.

FIG. 11 represents the absorbance at 230 nm of various fractions (F) of15 ml obtained during an enzymatic synthesis of heparin at degrees ofpolymerization dp ranging from 2 to 10 (the dp corresponds to thefigures indicated on the curve).

FIG. 12 is a graph produced from the experimental results of Example 10.This graph shows the percentages of gp120/CXCR4 interaction in thepresence or absence of heparin.

EXAMPLES

In the following examples, the analyses of gp120-heparin interactionwere carried out by surface plasmon resonance (BIAcore system(trademark)). This technique, which makes it possible to performreal-time interaction measurements, also has the advantage of providinga model similar to the physiological reality, where the heparinimmobilized on the sensorchip constitutes a two-dimensional interfacelike the cell surface.

Example 1 Synthesis of a CD4 Peptide which can be used for Producing theComposition of the Present Invention

A peptide from a sequence listing attached in the appendix issynthesized by solid-phase chemical synthesis with an Applied Biosystemsautomatic peptide synthesizer, mod. 433A, and by Fmoc chemistry, whichuses the fluorenylmethyloxycarbonyl (Fmoc) group for temporaryprotection of the α-amino function of the amino acids. The protectivegroups used to prevent the side reactions of the amino acid side chains,in this Fmoc strategy, were tert-butyl ether (tBu) for the Ser, Thr andTyr residues; tert-butyl ester (OtBu) for Asp, Glu; trityl (Trt) forGln, Asn, Cys, His; tert-butyloxycarbonyl (Boc) for Lys and2,2;5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for Arg.

The coupling reaction takes place with an excess of 10 equivalents ofamino acids (1 mmol) relative to the resin (0.1 mmol). The protectedamino acid is dissolved in 1 ml of N-methylpyrrolidone (NMP) and 1 ml ofa 1 M solution of 1-N-hydroxy-7-azabenzotriazole (HOAt) in the NMPsolvent. 1 ml of a 1 M solution of N,N′-dicyclohexylcarbodiimide (DCC)is then added. After activation for 40 to 50 minutes, the active esterformed is transferred into the reactor which contains the resin. Beforethis transfer then coupling step, the resin is deprotected by removal ofits Fmoc group with a 20% solution of piperidine in NMP. The excesspiperidine is removed by washing with NMP after approximately 5 to 10minutes.

After synthesis of the peptide, the peptide-resin is treated five timeswith a 2% solution of hydrazine in DMF. The coupling of a linker arm iscarried out for one hour at ambient temperature in DMF with 10equivalents of Fmoc-8-amino-3,6-dioxaoctanoic acid using the HBTUreagent in the presence of diisopropyl-ethylamine. The Fmoc group isthen deprotected with 20% of piperidine in DMF.

The peptide-resin is immediately treated with 10 equivalents of Trautreagent (2-iminothiolane hydrochloride (Sigma)) in the presence of DIEA.The peptide is finally released and deprotected as described below.

The cleavage of the resin and the cleavage of the protective groupspresent on the side chains were carried out simultaneously by treatingthe peptide linked to the resin with trifluoroacetic acid (TFA). Beforeperforming the cleavage, the resin was washed several times withdichloromethane (DCM) and finally dried. The reagent used during thecleavage is an acid mixture containing 81.5% of TFA and phenolscavengers (5%), thioanisol (5%), water (5%), ethanediol (2.5%) andtriisopropylsilane (1%). The resin was treated with this mixture forthree hours with stirring and at ambient temperature, in a proportion of100 ml of solution per gram of resin. The free peptide in solution wasrecovered by filtration. The peptide was then precipitated and washedunder cold conditions in diisopropyl ether and then dissolved in 20%acetic acid and lyophilized.

The peptide recovered after lyophilization, the synthesis crude, is inreduced form, i.e. the intrachain disulphide bridges are not formed. Theformation of these covalent bonds was performed using thecystamine/cysteamine redox couple. The synthesis crude was taken up inwater to which 0.1% (v/v) of TFA and 6 M guanidinium chloride had beenadded in order to facilitate the dissolving thereof, in a proportion of2.0 mg·ml⁻¹. This solution was then added, dropwise, diluted to 0.2mg/ml⁻¹, to the reducing buffer, made up of 100 mM Tris/HCl, pH 7.8, and5 mM cysteamine. Cystamine (oxidizing agent), at a final concentrationof 0.5 mM, was added after 45 minutes of reaction at ambienttemperature. The medium was brought to pH 3.0 after 30 minutes.

The cysteamine makes it possible to reduce the thiol groups present onthe peptide. In the open air, it oxidizes and allows the oxidation ofcysteines and therefore the folding of the peptide by formation ofintrachain disulphide bridges. The cystamine added at the end of themanipulation makes it possible to complete the folding. The correctprogress of the oxidation is verified by analytical chromatography,comparing the retention times of the crude and oxidized products, whichare greater for the former.

The peptides were purified by reverse-phase high performance liquidchromatography on a Vydac C18 (1.0×25.0 cm) preparative column. A lineargradient of 0-60% acetonitrile in a 0.1% aqueous trifluoroacetic acidsolution, over 90 minutes, was used. The fractions of the major peakwere analysed by analytical HPLC; the fractions exhibiting just one peakwere combined and lyophilized.

The products thus obtained were analysed by mass spectrometry. They arethe peptides of the sequence listing attached in the appendix.

Example 2 Synthesis of Polyanion of the Heparin or Heparan SulphateType, which can be used for the Composition of the Present Invention

A) Enzymatic Synthesis

A molecule of heparin or of heparan sulphate having a defined degree ofpolymerization dp is synthesized.

6 g of heparin are solubilized in a buffer containing 5 mM of Tris, 2 mMof CaCl₂, 50 mM of NaCl and 0.1 mg/ml of albumin. The pH is adjusted to7.5 with acetic acid. This solution is incubated at 25° C. withheparinase I (8 mU/ml) for approximately 50 h (the enzymatic reaction ismonitored by means of the increase in optical density, measured at 232nm).

The mixture is then purified by gel filtration chromatography. The solidphase is Biogel P10, contained in a 1.50 m column 4.4 cm in diameter,eluted at 1 ml/min with 0.25 M NaCl.

FIG. 11 represents the absorbance at 230 nm of the various fractions of15 ml obtained for degrees of polymerization dp ranging from 2 to 10.

The various oligosaccharides (dp2, dp4, etc.) are dialysed against waterand then lyophilized.

B) Synthesis by Chemical Depolymerization from the Natural Product

When the starting material is heparin, the following procedures arecarried out: 1 g of heparin is solubilized in 20 ml of sodium nitrite(NaNO₂) at 2.1 mg/ml. The solution is adjusted to pH 1.5 with sulphuricacid, and is then incubated at 4° C. for 3 h. The reaction is stoppedand the oligosaccharides are purified as above in paragraph A).

When the starting material is heparan sulphate, the following procedureis carried out: 8 g of heparan sulphate are solubilized in 40 mlcontaining 5 mM of Tris, 2 mM of CaCl₂, 50 mM of NaCl and 0.1 mg/ml ofalbumin. The pH is adjusted to 7.5 with acetic acid. This solution isincubated at 30° C. with heparinase III (25 mU/ml) for approximately 72h. Heparinase III is again added, for a period of 48 h, and the productsare then purified as described above in paragraph A).

Example 3 Synthesis of a Composition of the Present Invention: Mixtureof a CD4 Peptide with a Polyanion

In this example, a CD4 peptide of Example 1 is mixed with a heparansulphate prepared in Example 2.

These two molecules are dissolved at a concentration that is two timesthe desired final concentration. These solubilizations are carried outin a physiological buffer; for example, PBS, TBS (50 mM Tris, 0.15 MNaCl, pH 7.5) or HBS (20 mM Hepes, 0.15 M NaCl, pH 7.5).

The two preparations are then mixed volume for volume (1/1).

Example 4 Synthesis of a Composition of the Present Invention: Couplingof a CD4 Peptide with a Polyanion

In this example, a CD4 peptide of Example 1 is coupled with the heparansulphate prepared in Example 2.

The heparan sulphate is incubated with a molar excess of hydrazine or ofcarbodihydrazide. The function of this step is to place a hydrazinegroup on the reductive end of the oligosaccharide, when it is preparedby enzymatic depolymerization, or on the aldehyde of theoligosaccharide, when it is prepared by chemical depolymerization withnitrous acid.

The carbohydrates of the soluble CD4 peptide are oxidized by treatmentwith sodium periodate, and the aldehyde function thus created is usedfor the coupling of the hydrazine-containing oligosaccharide.

The oligosaccharide, generally in solution at 1 mM in PBS buffer (sodiumphosphate saline), is coincubated with a molar excess (for example up to100 times) of hydrazine or of carbodihydrazide, also in solution in PBS.The reaction mixture is incubated at ambient temperature, then purifiedby desalification or dialysis against distilled water and, finally,dried by evaporation under vacuum or lyophilized.

The glycosylated (produced in mammalian cells or insect cells) solubleCD4 molecule (sCD4) is taken up in a 20 mM phosphate buffer, pH 6.2, andthen treated with sodium periodate (10 mM) for 20 minutes at 4° C. andin the dark. To remove the sodium periodate, the reaction mixture isdesalified by gel filtration or by dialysis against the phosphatebuffer.

The sCD4, the glycans of which are thus oxidized, is coincubated with amolar excess of hydrazine-containing oligosaccharide at 4° C., so as toform a complex between the two molecules.

When the CD4 is not glycosylated, the procedure is carried out in themanner described by Najjam et al., in document [17].

It is also possible to use any type of bridging agent which binds,firstly, to one end of the oligosaccharide and, secondly, to a part ofthe CD4 peptide that is not essential to its function. Those skilled inthe art will have no difficulty in implementing this process or anequivalent process.

Example 5 Demonstration of the Increase in the Affinity of gp120 forHeparin by Means of CD4

30 resonance units (RU) of biotinylated heparin are immobilized at thesurface of a biochip (“sensorchip” B1 produced by the company Biacore).

gp120 (hxbc2) at 50 nM is incubated for 1 hour 30 min with increasingconcentrations of soluble CD4 at 0, 50, 100, 250 or 500 nM, and theninjected onto the heparin surface at 10 μl/min.

The analyses of gp120-heparin interaction by surface plasmon resonancewere carried out as a function of time.

The curves in FIG. 1 correspond to the injection of gp120 at 50 nM andof CD4 respectively at 0, 50, 100, 250 or 500 nM (respectively for thecurves from bottom to top in this figure).

FIG. 2 shows the amount of gp120/CD4 bound to the heparin as a functionof the concentration of CD4.

It appears that a CD4:gp120 molar ratio of approximately 5:1 producesthe maximum response.

These results show that exposure of the CD4i domain of gp120 greatlyincreases the interaction of gp120 with heparin. CD4i thereforerepresents a new site of interaction with heparin.

Example 6 CD4-Dependent gp120/48D Interaction

1250 RU of antibody 48d specific for the CD4i epitope are immobilized atthe surface of a biochip (“sensorchip” B1) as in Example 1 above.

The gp120 viral protein (hxbc2) at 50 nM is incubated for 1 h 20 minwith increasing concentrations of soluble CD4 at 0, 50, 100, 240 or 500nM, and then injected onto the 48d surface at 10 μl/min.

The analyses of gp120-heparin interaction by surface plasmon resonancewere carried out as a function of time.

The curves in FIG. 3 correspond to the injection of gp120 at 50 mM andof CD4 respectively at 0, 50, 100, 250 or 500 nM (respectively for thecurves from bottom to top in this figure).

This example shows that the gp120/48d interaction is CD4-dependent, andthat 48d interacts with CD4i, the coreceptor recognition domain. Thisantibody can therefore be used as a model for the interaction of gp120with a coreceptor.

Example 7 Inhibition of the gp120 Protein-48d Interaction by Heparin

The gp120 protein is coincubated for 40 minutes with CD4. The mixture isthen divided up into 5 aliquots, to which the heparin (15 kDa) is addedat various concentrations.

The final concentrations in the aliquots are: gp120:50 nM; CD4:250 nMand heparin: 0, 3, 6, 12 or 30 μg/ml, respectively, from top to bottomin FIG. 4. On the top curve, where there is no heparin, the gp120/CD4interaction is visualized; all the other curves are in the presence ofheparin (from 3 to 30 μg/ml, respectively, from top to bottom, FIG. 4).

After incubation for 40 minutes, the various mixtures are injected ontothe 48d surface.

The analyses of gp120-heparin interaction by surface plasmon resonancewere carried out as a function of time.

The results obtained are represented in FIG. 4. They show that heparininhibits the adsorption of the gp120/CD4 complex onto the 48d-antibodysurface. The heparin is, moreover, found to be an effective inhibitorsince the inhibition is virtually complete from the lowest of theconcentrations tested (3 μg/ml).

This shows that the heparin competes with 48d and therefore binds toCD4i.

This example indicates that the inhibitory activity of theoligosaccharides (heparin), as defined above, is obtained in thepresence of CD4.

This result makes it possible to propose the use of a hybrid moleculemade up of CD4 and of oligosaccharides of the heparin type, linkedcovalently, or of a mixture of these two molecules.

The direct interaction of the CD4i domain with a polyanion has neverbeen described in the prior art, neither has inhibition of thegp120-antibody 48d interaction by a polyanion. No studies existedshowing the possible inhibition of gp120 with the coreceptors by meansof a molecule of the heparin type.

Example 8 Inhibition of the gp120-48d Interaction with OligosaccharideFragments of Defined Sizes

The oligosaccharide fragments of defined sizes are obtained by enzymaticdepolymerization.

The gp120 viral protein is coincubated for 60 minutes with CD4 so as toexpose the CD4i domain. The mixture is divided up into 8 aliquots andheparin fragments of increasing size, comprising from 1 to 8 basicdisaccharide units, i.e. a degree of polymerization (dp) of 2 to 16, areadded so as to give final concentrations of 50 nM for gp120, 250 nM forCD4 and 125 nM for the heparin fragments (the molecular mass of adisaccharide is approximately 600 Da).

The mixtures are then injected onto the 48d surface (FIG. 5). The dataobtained make it possible to represent the amount of gp120/CD4 complexesbound to 48d as a function of the size of the heparin fragment (FIG. 6).

These results show that oligosaccharides of size equal to or less thandp6 (3 disaccharides) do not have the ability to block the interactionof the gp120/CD4 complex with 48d.

On the other hand, the interaction is completely inhibited by thefragments whose size is greater than dp10, and better still greater thandp12, at the concentration tested.

These results reveal, for example, that a concentration of 125 nM ofheparin having a degree of polymerization equal to 16 (dp16), i.e. 0.6μg/ml, in the presence of CD4 at 250 nM, inhibits 90 to 100% of theinteraction of the 48d antibody with gp120 (50 nM), which confirms theexistence of a direct interaction. Similarly, oligosaccharides ofheparin of type dp14 to dp18 at 40 nM inhibit the interaction of thegp120 (5 nM)/CD4 (10 nM) complex with 17b antibody.

These results show that heparin, a molecule having a structure verysimilar to heparan sulphates, also interacts with a second binding siteon gp120. This site, called CD4i, is an epitope that is only exposedwhen gp120 interacts with CD4, which constitutes the HIV coreceptorbinding site. These results show that the presence of CD4, the effect ofwhich is to expose the CD4i site, very substantially increases thegp120-heparin or gp120-heparan sulphate interaction; this has never beendescribed elsewhere and constitutes the first proof of a possibleinteraction between gp120 and heparin or heparan sulphate via the CD4isite.

A molecular modelling study showed that the CD4i site of gp120 consistsof basic amino acids. These basic amino acids are aligned on the surfaceof the protein, and effectively constitute a site for interaction withheparin, or oligosaccharides derived from heparin or from heparansulphate.

The inventors therefore hereby propose a therapeutic use of thepolyanionic compounds targeting this novel site of interaction. Theapproach consists of the conjugated use of polyanions and of moleculescapable of exposing the CD4i epitope, by coadministration or in the formof a hybrid molecule. This type of molecule simultaneously blocks allthe domains of interaction of gp120 with the host cells.

Example 9 Protocol for the Activation of Oligosaccharides for thePurpose of Coupling them with a Protein

The protocol is based on the reaction of an aldehyde group (on theoligosaccharide) with an amine or hydrazide group on the protein.

When the oligosaccharides are obtained by chemical depolymerization ofheparin (with nitrous acid), the aldehyde function is created at thecleavage site and the oligosaccharide is ready for the coupling.

When the oligosaccharides are obtained by enzymatic depolymerization,the following procedure may be carried out:

The oligosaccharides, at a concentration of approximately 10 mM, areincubated in a saturating solution of ammonium bicarbonate for 96 hours.The reaction mixture is then purified on a gel filtration column,equilibrated with 10 mM ammonium bicarbonate, and the sample is thenlyophilized several times in order to eliminate the residual ammoniumbicarbonate. The aim of this step is to create a glycosylamine at thereductive end of the oligosaccharide.

Alternatively, the oligosaccharide (10 mM) may also be incubated with0.25 mM of dihydrazide adipate, in the presence of sodiumcyanoborohydride (NaBH₃CN, 1 M) at pH 5, for 96 hours. The reactionmixture is then purified on a gel filtration column, equilibrated indistilled water, and then dried by lyophilization. The aim of this stepis to introduce a hydrazide function at the reductive end of theoligosaccharide.

The oligosaccharides, prepared according to the two methods above, arethen incubated with 0.5 M of diglutyraldehyde, at pH 5, for 4 hours, andthen with 0.1 M of NaBH₃CN for 30 minutes. The reaction mixture is thenpurified on a gel filtration column, equilibrated in distilled water,and then dried by lyophilization. The aim of this step is to place analdehyde function at the reductive end of the oligosaccharides.

Example 10 Demonstration of the Inhibitory Activity of theOligosaccharides on the Interaction of GP120 with the Coreceptor (CXCR4)for the Virus

CHO cells (mutant 2241, deficient in glycosaminoglycan expression, theydo not produce heparan sulphate) transfected with the CXCR4 gene arepreincubated with: gp120 (20 μg/ml), with or without heparin, orgp120/CD4 complexes (20 μg/ml for each of the proteins), or gp120/CD4complexes incubated beforehand with 6 kDa heparin (10 μg/ml) or aheparin dodecasaccharide (10 μg/ml).

The gp120 bound to the surface of the cells is detected using ananti-gp120 antibody coupled to an FITC-labelled secondary antibody, andthen analysed by FACS.

The results represented in FIG. 12 attached in the appendix show thepercentages of gp120/CXCR4 interaction. The negative control (0%)corresponds to the non-specific binding of gp120 to the cells (gp120alone or in the presence of heparin) (1). The positive control (100%) isobserved for the gp120/CD4 complexes (2), and corresponds to the bindingof gp120 to the CXCR4 coreceptor, induced by CD4. The heparin (3) andthe dodecasaccharide (4) both greatly decrease the interaction (8.5 and4.1%, respectively, i.e. 91.5 and 95.9% inhibition).

The heparin used clearly inhibits the interaction of gp120 with thecoreceptor.

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1-18. (canceled)
 19. A composition comprising: a polyanion selected fromthe group consisting of heparin, heparan sulphate, a polyanionequivalent to heparin and a polyanion equivalent to heparan sulphate,said polyanion having a degree of polymerization dp of 10 to 24, and aCD4 peptide of sequence (1) below: Cys or TPA-P-Cys-P²-Cys-P³-Cys-Ala orGln-Gly or (D)Asp or Ser-Ser or His or Asn-Xaa^(J)-Cys-Thr orAla-Cys-Xaa^(k)-NH₂ wherein TPA represents thiopropionic acid, Xaa^(J)represents β-naphthylalanine, phenylalanine or biphenylalanine, Xaa^(k)represents Gly, Val or Ileu, P¹ represents 3 to 6 amino acids, P²represents 2 to 4 amino acids and P³ represents 6 to 10 amino acids, theamino acids in P¹, P² and P³ being natural or unnatural, identical ordifferent, and P¹, P² and P³ optionally having a common sequence, saidpeptide having a β-hairpin conformation wherein the β-turn comprises theamino acid residues Ala or Gln-Gly or DAsp or Ser-Ser or His orAsn-Xaa^(J) of its sequence (A).
 20. The composition according to claim19, wherein said polyanion has a degree of polymerization dp of 12 to20.
 21. The composition according to claim 19, wherein said polyanionhas a degree of polymerization dp of 15 to
 17. 22. The compositionaccording to claim 19, wherein the CD4 peptide of sequence (I) isselected from group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. 23.The composition according to claim 19, wherein the composition comprises1 to 10 mol of polyanion per 0.5 to 1.5 mol of the CD4 peptide.
 24. Thecomposition according to claim 19, wherein the composition comprises 5mol of polyanion per mole of the CD4 peptide.
 25. An anti-HIVcomposition comprising the composition according to claim 19.